Eliana Noelia Alonso

Maitake (D Fraction) Mushroom Extract Induces Apoptosis in Breast Cancer Cells by BAK-1 Gene Activation

For many years mushrooms have been used empirically in traditional medicine to treat several diseases. Study of the maitake mushroom, with its immunomodulatory and antitumoral properties, has led to the isolation of several bioactive compounds. One of these, D fraction, is known to reduce tumor cell viability. This study examined the effect of isolated D fraction on viability and apoptosis of human breast cancer cells (MCF7). These cells were treated with maitake (D fraction) extract at 18 μg/mL, 36 μg/mL, 91 μg/mL, 183 μg/mL, or 367 μg/mL or were left untreated (control) for 24 hours. MCF7 incubation with the maitake extract resulted in decreased cell viability [3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium assay] in a dose-dependent manner. Apoptosis was statistically significantly increased in a dose-dependent manner at every concentration tested (terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling assay). Upon incubation with D fraction, a microarray assay revealed upregulation of BAK-1 and cytochrome c transcripts, 2 proteins directly involved in the apoptotic pathway. Reverse transcriptase polymerase chain reaction studies confirmed these findings; BAK-1 was one of most overexpressed gene, as observed by microarray assay. These findings confirm the apoptotic effect of maitake D fraction in breast cancer cells and further highlight the involvement of cytochrome c release to the cytoplasm. Cytoplasmic release of cytochrome c, another player in the apoptotic pathway, was also increased after incubation with D fraction in a dose-dependent manner. This finding indicates that the effect of this compound involves mitochondrial dysfunction. The identification of the molecular mechanisms by which D fraction exerts its effects is crucial for the development of preventive and therapeutic strategies for cancer.

Structure-anti-leukemic activity relationship study of ortho-dihydroxycoumarins in U-937 cells: key role of the δ-lactone ring in determining differentiation-inducing potency and selective pro-apoptotic action.

Previous studies indicated the need of at least one phenolic hydroxyl group in the coumarin core for induction of cytotoxicity in different cell lines. Herein, we present an exhaustive structure-activity relationship study including ortho-dihydroxycoumarins (o-DHC) derivatives, cinnamic acid derivatives (as open-chain coumarin analogues) and 1,2-pyrones (representative of the δ-lactone ring of the coumarin core), carried out to further identify the structural features of o-DHC required to induce leukemic cell differentiation and apoptosis in U-937 cells. Our results show for the first time that the δ-lactone ring positively influences the aforementioned biological effects, by conferring greater potency to compounds with an intact coumarin nucleus. Most tellingly, we reveal herein the crucial role of this molecular portion in determining the selective toxicity that o-DHC show for leukemic cells over normal blood cells. From a pharmacological perspective, our findings point out that o-DHC may be useful prototypes for the development of novel chemotherapeutic agents.

The alkynylphosphonate analogue of calcitriol EM1 has potent anti-metastatic effects in breast cancer

The active form of vitamin D3, calcitriol, plays a major role in maintaining calcium/phosphate homeostasis. In addition, it is a potent antiproliferative and prodifferentiating agent. However, when effective antitumor doses of calcitriol are employed, hypercalcemic effects are observed, thus precluding its therapeutic application. To overcome this problem, structural analogues have been designed with the aim at retaining or even increasing the antitumor effects while decreasing its calcemic activity. This report shows the biological evaluation of an alkynylphosphonate vitamin D less-calcemic analogue in a murine model of breast cancer. We demonstrate that this compound has potent anti-metastatic effects through its action over cellular migration and invasion likely mediated through the up-regulation of E-cadherin expression. Based on the current in vitro and in vivo results, EM1 is a promising candidate as a therapeutic agent in breast cancer.

Genes Related to Suppression of Malignant Phenotype Induced by Maitake D-Fraction in Breast Cancer Cells

It is already known that the Maitake (D-Fraction) mushroom is involved in stimulating the immune system and activating certain cells that attack cancer, including macrophages, T-cells, and natural killer cells. According to the U.S. National Cancer Institute, polysaccharide complexes present in Maitake mushrooms appear to have significant anticancer activity. However, the exact molecular mechanism of the Maitake antitumoral effect is still unclear. Previously, we have reported that Maitake (D-Fraction) induces apoptosis in breast cancer cells by activation of BCL2-antagonist/killer 1 (BAK1) gene expression. At the present work, we are identifying which genes are responsible for the suppression of the tumoral phenotype mechanism induced by Maitake (D-Fraction) in breast cancer cells. Human breast cancer MCF-7 cells were treated with and without increased concentrations of Maitake D-Fraction (36, 91, 183, 367 μg/mL) for 24 h. Total RNA were isolated and cDNA microarrays were hybridized containing 25,000 human genes. Employing the cDNA microarray analysis, we found that Maitake D-Fraction modified the expression of 4068 genes (2420 were upmodulated and 1648 were downmodulated) in MCF-7 breast cancer cells in a dose-dependent manner during 24 h of treatment. The present data shows that Maitake D-Fraction suppresses the breast tumoral phenotype through a putative molecular mechanism modifying the expression of certain genes (such as IGFBP-7, ITGA2, ICAM3, SOD2, CAV-1, Cul-3, NRF2, Cycline E, ST7, and SPARC) that are involved in apoptosis stimulation, inhibition of cell growth and proliferation, cell cycle arrest, blocking migration and metastasis of tumoral cells, and inducing multidrug sensitivity. Altogether, these results suggest that Maitake D-Fraction could be a potential new target for breast cancer chemoprevention and treatment.

The UVB1 Vitamin D analogue inhibits colorectal carcinoma progression.

Vitamin D has been shown to display a wide variety of antitumour effects, but their therapeutic use is limited by its severe side effects. We have designed and synthesized a Gemini vitamin D analogue of calcitriol (UVB1) which has shown to display antineoplastic effects on different cancer cell lines without causing hypercalcemia. The aim of this work has been to investigate, by employing in silico, in vitro, and in vivo assays, whether UVB1 inhibits human colorectal carcinoma progression. We demonstrated that UVB1 induces apoptotic cell death and retards cellular migration and invasion of HCT116 colorectal carcinoma cells. Moreover, the analogue reduced the tumour volume in vivo, and modulated the expression of Bax, E-cadherin and nuclear β-catenin in tumour animal tissues without producing toxic effects. In silico analysis showed that UVB1 exhibits greater affinity for the ligand binding domain of vitamin D receptor than calcitriol, and that several characteristics in the three-dimensional conformation of VDR may influence the biological effects. These results demonstrate that the Gemini vitamin D analogue affects the growth of the colorectal cancer and suggest that UVB1 is a potential chemotherapeutic agent for treatment of this disease.

Maitake Pro4X has anti-cancer activity and prevents oncogenesis in BALBc mice

The understanding of the molecular mechanisms of the immune tolerance induced by the tumoral microenvironment is fundamental to prevent cancer development or to treat cancer patients using immunotherapy. Actually, there are investigations about “addressed‐drugs” against cancer cells without affecting normal cells. It could be ideal to find selective and specific compounds that only recognize and destroy tumor cells without damaging the host normal cells. For thousands of years, mushrooms have been used for medicinal purposes because of their curative properties. D‐Fraction, an extract of Maitake (from the edible Grifola frondosa mushroom), rich in β‐glucans, exert notable effects in the immune system. Until now, some published articles suggest that Maitake D‐Fraction could have anti‐tumoral activity, prevent oncogenesis and metastasis in some tumor types. However, there are no clear data about Maitake D‐Fraction action on breast cancer prevention and its exact molecular mechanisms are not yet elucidated. The experiments were performed employing 25 female BALBc mice that were treated with and without Maitake D‐Fraction Pro4X or Maitake Standard for 15 days by daily intraperitoneal injection. After treatment period, all mice were implanted with murine tumor cells LM3 to induce mammary tumorigenesis. Animals were checked weekly and killed after 46 days of LM3 transplant; percentage of cancer prevention, rate of tumor growing, and overall survival were determined. Under dissection, the internal organs were evaluated histologically and genetically by RT‐PCR. We found that 5 mg/kg per day of Maitake D‐Fraction Pro4X, administered dairy during 15 days to BALBc mice was able to block more than 60% breast cancer development. However, Maitake Standard prevents oncogenesis in 26% to respect control. In this work, we found that Maitake D‐Fraction Pro4X, administered to BALBc mice, prevents breast carcinogenesis, block tumor invasiveness, reduce angiogenesis, and increase overall survival.

BRCA1 polymorphism in breast cancer patients from Argentina

Breast cancer is the most common type of cancer in females in Argentina, with an incidence rate similar to that in the USA. However, the contribution of the BRCA1 or BRCA2 mutation in breast cancer incidence has not yet been investigated in Argentina. In order to evaluate which BRCA1 polymorphisms or mutations characterize female breast cancer in Argentina, the current study enrolled 206 females with breast cancer from several hospitals from the southeast of Argentina. A buccal smear sample was obtained in duplicate from each patient and the DNA samples were processed for polymorphism analysis using the single-strand conformational polymorphism technique. The polymorphisms in BRCA1 were investigated using a combination of 15 primers to analyze exons 2, 3, 5, 20 and 11 (including the 11.1 to 11.12 regions). The BRCA1 mutations were confirmed by direct sequencing. Samples were successfully examined from 154 females and, among these, 16 mutations were identified in the BRCA1 gene representing 13.9% of the samples analyzed. One patient was identified with a polymorphism in exon 2 (0.86%), four in exon 20 (3.48%), four in exon 11.3 (3.48%), one in exon 11.7 (0.86%), two in exon 11.8 (1.74%), one in exon 11.10 (0.86%) and one in exon 11.11 (0.86%). The most prevalent alteration in BRCA1 was located in exon 11 (11 out of 16 patients; 68.75%). The objective of our next study is to evaluate the prevalence of mutations in the BRCA2 gene and analyze the BRCA1 gene in the healthy relatives of BRCA1 mutation carriers.

Antitumoral and antimetastatic activity of Maitake D-Fraction in triple-negative breast cancer cells

Triple-negative breast cancer (TNBC) is associated with poor prognosis, high local recurrence rate and high rate of metastasis compared with other breast cancer subtypes. In addition, TNBC lacks a targeted therapy. This scenario highlights the need for novel compounds with high potential for TNBC treatment. In this regard, natural products are important sources of anticancer drugs. D-Fraction, a proteoglucan extracted from the edible and medicinal mushroom Grifola frondosa (Maitake), is a dietary supplement that has been shown to exert both immunostimulatory and immune-independent antitumoral effects on some cancer types. However, its antitumoral potential in TNBC is unknown. Therefore, we employed TNBC cells to investigate if D-Fraction is able to attenuate their aggressive phenotype. We found that D-Fraction decreases MDA-MB-231 cell viability through apoptosis induction and reduces their metastatic potential. D-Fraction increases cell-cell adhesion by increasing E-cadherin protein levels and β-catenin membrane localization, and increases cell-substrate adhesion. D-Fraction also decreases cell motility by affecting actin cytoskeleton rearrangements, and proteolytic activity of MMP-2 and MMP-9. Furthermore, D-Fraction decreases the invasive capacity of MDA-MB-231 cells. In concordance, D-Fraction retards tumor growth and reduces lung metastases in a xenograft model. Altogether, these results suggest the potential therapeutic role of D-Fraction in aggressive TNBC.

Abstract B41: A NUTRIGENOMIC AGENT INDUCES BREAST CANCER PREVENTION IN BALBc MICE.

Nutrigenomics is the scientific discipline that explains how nutrition can influence the switching on and off of genes and improve health outcomes. Is already known that eating healthy food helps our bodies get the needed nutrients for strengthening the immune system and fighting against illness. Many websites discuss using mushrooms to treat cancer. It has been reported that Maitake D-Fraction exert its antitumor effect in tumor-bearing mice by enhancing the immune system through activation of macrophages, T cells, and natural killer (NK) cells. The specific proteo-glucan in Maitake mushroom extract for fighting cancer tumors is called the D-fraction. Maitake-D-fraction has been reported to exert its antitumor effect in tumor-bearing mice by enhancing the immune system through activation of macrophages, T cells, and natural killer (NK) cells. The proteo-glucan shows anticarcinogenic activity, prevent oncogenesis and prevent metastasis. However the exact molecular mechanisms and gene expression generated by Maitake-D-fraction in the anti-carcinogenesis process are still unclear. We have been reported that D-fraction Standard (Mushrooom Wisdom, Inc., NJ, USA.) induces apoptosis in breast cancer cells by activation of BAK1 gene expression; we also found that cytochrome C1 was released to the cytoplasm in a dose-dependent manner. Recently we identified certain genes responsible for the suppression of the tumoral phenotype induced by Maitake-D-fraction in breast cancer cells. In the present work we are developing the in-vivo experiments studying whether Maitake-D-Fraction can prevent breast cancer development in BALBc female mice. We did perform three groups employing 5 BALBc female mice (6 weeks old) in each one; in the Control group we use 6 animals. Group 1 is control (treated by i.p injection with 100μl of PBS), Group 2 treated by i.p injection with 5mg/kg/day of Maitake D fraction Standard and Group 3 treated by i.p injection with 5mg/kg/day of Maitake Pro 4X (Mushroom Wisdom, NJ, USA) during 15 days. After that time, we did induce breast tumorigenesis by i.p. injection of 4x105 LM3 murine tumoral cells. Each animal were daily controlled for breast tumor by palpation and measurement. 6 out 6 animals in the control group 1 develop breast tumor after 10 days of tumor cells injection (100% of tumor development). 2 out of 5 animals (40%) develop tumor in the Group 2 treated with Standard Maitake after 12 days of tumor cells injection and none (0 out of 5 animals) develop tumor in the Group 3 treated with Maitake Pro 4X. Meaning that 5mg/kg/Day of Maitake D-Fraction Pro 4X prevent 100% of breast tumorigenesis in BALBc mice after 15 days. In Conclusion, understanding the molecular mechanisms of D-fraction Pro 4X may lead to the development of a new nutrigenomic agent able to generate efficient prevention for breast cancer development in high risk patients. Citation Format: Eliana Noelia Alonso, Diego Javier Obiol, Gabriela Andrea Balogh. A nutrigenomic agent induces breast cancer prevention in BALBc mice. [abstract]. In: Proceedings of the Eleventh Annual AACR International Conference on Frontiers in Cancer Prevention Research; 2012 Oct 16-19; Anaheim, CA. Philadelphia (PA): AACR; Cancer Prev Res 2012;5(11 Suppl):Abstract nr B41.

Abstract 3262: Pharmaco-modulation of dihydroxy coumarins towards more selective antileukemic agents

Background: We described that 7,8-dihydroxy-4-methylcoumarin (DHMC) induces apoptosis in human leukemic U-937 cells, mediated by its pro-oxidant activity and activation of JNK. We also reported that DHMC shows selective toxicity for leukemic cells over normal peripheral blood monocytes. The aim of the present work is to establish the structural requirements for DHMC to display pro-apoptotic activity performing a structure activity relationship study of DHMC derivatives, cinnamic acid derivatives (as open-chain coumarin analogues) and a-pyrones (as representative of the d-lactone ring of the coumarin nucleus). Further, in an attempt to explain its selective toxicity, the mechanism of DHMC action through the JNK pathway will be characterized. Materials and Methods: DHMC analogues, cinnamic acid derivatives and a-pyrones were tested for their ability to induce biological effects in U-937 cells. Growth inhibition was determined through 3H-thymidine incorporation. Cellular viability was tested by trypan blue exclusion assays. Pro-apoptotic activity was evaluated by flow cytometry (annexin V), determination of caspase-3 activity and Western Blot analysis of caspase-3 and cleaved PARP levels. The oxidation potential and the capacity to generate free radicals were evaluated by cyclic voltammetry and electron paramagnetic resonance, respectively. To evaluate the role of cytoplasmic p21 (Cip1/WAF1) in the mechanism of DHMC selective action, we used U-937 cells transfected with a zinc-inducible p21 deletion mutant lacking the nuclear localization signal. Results: Only DHMC derivatives bearing two adjacent aromatic hydroxyl groups (catechol moiety) showed proliferation inhibition and pro-apoptotic activity in U-937 cells. a-pyrones had no effect on cellular viability, and cinnamic acid derivatives bearing the catechol moiety exhibited proliferation inhibition but no cytotoxic activity. Although ortho-dihydroxy coumarins and ortho-dihydroxy cinnamic acid derivatives showed a similar oxidation potential, only the former could generate stable free radicals in medium-like conditions, suggesting this capacity would be critical for their pro-apoptotic activity in U-937 cells. Finally, the pro-apoptotic effect of DHMC via JNK activation was abolished in U-937 cells expressing cytoplasmic p21, indicating this protein would be involved in the resistance that normal lymphocytes and monocytes, which normally express p21 in their cytoplasm, show for DHMC. Conclusions: DHMC-derived compounds carrying a catechol moiety can only induce apoptosis in U-937 cells if the integrity of the coumarinic nucleus is preserved. Selective toxicity of DHMC for leukemic cells would involve activation of JNK in the absence of cytoplasmic p21 expression. Collectively, our results point to ortho-dihydroxy coumarins as promising prototypes for the design of more selective antileukemic drugs. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3262. doi:10.1158/1538-7445.AM2011-3262